This application requests funds to establish a Biotechnology Research Resource devoted to pulsated EPR spectroscopy. The instrument to be supported is a unique, wide band pulsed EPR spectrometer, operating from 8-18 GHz and specifically designed for electron spin echo (ESE) spectroscopy of biological materials. Core development associated with the resource includes implementation of spin echo ENDOR spectroscopy as an alternative to conventional continuous wave ENDOR methods. Pulsed ENDOR methods are inherently more sensitive and can be more readily applied to the study of magnetically dilute biological materials. A second aspect of core development is new cavity utilization and design for ESE spectroscopy to achieve greater instrument sensitivity. Core research initially will be devoted to the study of 14N hyperfine interactions in hemoproteins and porphyrins, addressing the relationship between tertiary and quaternary structure and magnetic coupling. Studies of mixed state nitrosyl and methomoglobins, and mixed metal (Fe, Co) hemoglobins are proposed. Other experiments involve comparisons of low spin forms of myoglobins and peroxidases which contain the same prosthetic groups and axial ligands, but with different crystal fields. In addition, the effect of relative orientations of axial heme ligands on magnetic interactions in a series of hemoproteins and model compounds of known structure will be addressed. A number of collaborative studies are proposed to examine electron-nuclear interactions using ESE and spin echo ENDOR methods. Here, experiments will be performed to identify the lignad composition of metal containing biomolecules and the proximity of substrates to metal centers in metalloenzymes. Other studies include the examination of non-centrosymmetric sites using the linear electric field effect (LEFE) in EPR. Service and educationsl activities related to pulsed EPR as a probe of the structure of paramagnetic biomolecules are proposed.